Critical nuclear DNA size and distribution associated with S phase initiation

Using HeLa S-3 cells synchronized by selective detachment, in this paper we report a parallel study of nuclear morphology and autoradiography grain patterns between middle G₁ and middle S phases: Our results show two distinct [³H]-thymidine labeling patterns. The first “peripheral” labeling pattern has a characteristic nuclear size distribution, in contrast to the heterogeneous and varying size distributions of Feulgen-stained nuclei, and apparently is characteristic of very early S phase. The sizes of the second labeling pattern—homogeneous or inhomogeneous grain distribution throughout the nucleus—are equal or larger than the first and vary with S phase progression. Together, the corresponding nuclear sizes of the labeled nuclei represent the larger extreme of nuclear areas, and the labeling index closely parallels the fraction of nuclei with areas larger than the minimum size of the labeled nuclei. These results suggest a characteristic nuclear size (reflecting unique intranuclear DNA distribution) as a necessary, if not sufficient, requirement for S phase initiation. Parallel experimentation with rat liver cells—synchronized in vivo by partial hepatectomy and analyzed by thin section autoradiography—confirms the existence of a peripheral labeling pattern in both the very early part and the very late part of S phase, which reconciles our data with previous results and points to the fact that both initiation and termination sites for DNA replication are near the nuclear periphery.     

Physico-chemical model for DNA alkaline elution: New experimental evidence and differential role of DNA length,chain flexibility and superpacking

For a better understanding of data provided by DNA alkaline elution technique, a new analytical model has been developed which takes into consideration both the physicochemical properties of in situ DNA strand (length and flexibility/superpacking) and the geometric and hydrodynamic configuration of the elution apparatus (flow and filter conditions). Simulation by this model of experimental data previously obtained before and after carcinogens administration, has shown that for constant flow and filter conditions elution profiles are dependent, not only from DNA molecular weight, but also from a parameter critically related to modifications in chain flexibility/superpacking. This has been confirmed by several independent observations, including the time-dependent changes in non-denaturing lysing solution monitored by hydroxylapatite and alkaline elution techniques.     

Differential scattering of circularly polarized light as a unique probe of polynucleosome superstructures. A simulation by multiple scattering of dipoles

A theoretical approach to modeling Circular Intensity Differential Scattering (CIDS) of native chromatin as multiple scattering of dipoles is discussed without the Born approximation. The model can explain the experimental data in the literature. It is shown that CIDS contains more structural information than does total light scattering and to a good approximation is independent of the length of the scattering molecules. Finally, CIDS in conjunction with traditional light scattering measurements should aid in discriminating between various alternative models of higher order chromatin structure now being proposed. Generalization of this theoretical study to other complex biomolecular structures, is also briefly discussed.     

Improvement of protein secondary structure prediction by combination of statistical algorithms and circular dichroism.

Three different approaches (propensity curve shifting, hydropathy index evaluation, and iterative attribution/cancellation of secondary structure) to the use of secondary structure percentages derived from circular dichroism measurements to improve the success rate of a protein secondary structure prediction method, without using decision constants, are described and compared. Propensity-curve shifting appears to be the best-performing approach, bearing an increase of 5.3% in the success rate of single-residue structural prediction when exact information on the secondary structure, obtained by X-ray crystallography, is employed; with information of an accuracy comparable to that obtainable by circular dichroism, the improvement stays between 3.5 and 4.9%, for a three-state prediction. Although developed with circular dichroism in mind, the method can use percentages of secondary structure obtained by any other experimental methodology from which they can be inferred, for instance Raman spectroscopy and infrared spectroscopy.     

Prenatal treatment of congenital human cytomegalovirus infection by fetal intravascular administration of ganciclovir

Ganciclovir was administered 'in utero' for 12 days in a 29-week-old fetus with ascertained congenital human cytomegalovirus (HCMV) infection, thrombocytopenia and elevated gamma-glutamyl transferase (gammaGT) value. Efficacy of therapy was shown by reduction in virus titer of amniotic fluid and fetal urine, disappearance of viral DNA in fetal blood, and normalization of platelet count and gammaGT value. However, stillbirth occurred at 32 weeks of gestation and HCMV inclusion bodies were detected in kidneys, lungs, heart and pancreas at autopsy. During therapy, side-effects,possibly related to ganciclovir administration, were observed.     

Characterization of Langmuir-Blodgett films of rhodopsin: thermal stability studies.

Two-dimensional close packing of purified bovine rhodopsin, made by the Langmuir-Blodgett technique, was characterized by small angle x-ray scattering and nanogravimetric measurements. The area occupied by a molecule of rhodopsin in the film was approximately 1100 Angstrum2 and the periodicity of the layers resulted in 59 Angstrum. The circular dichroism measurements showed that bleached rhodopsin in Langmuir-Blodgett film had high thermal stability, in fact, reaching a temperature of 150 degrees C without a loss of the secondary structure. Moreover, when the film was made up in the dark, rhodopsin maintained its stability up to at least 200 degrees C and its characteristic absorbance peak at 500 nm up to about 90 degrees C.     

Reversible (G0) and nonreadily reversible (Q) noncycling cells in human peripheral blood. Immunological, structural, and biological characterization

PHA-stimulated human lymphocytes (normal-resting-proliferating) at 0, 24, 48, 72, and 144 h were studied with Acridine Orange (AO) staining. By viable cell sorting, by subsequent subculturing, and by the use of biochemical, biophysical, and immunological assays, not only have the G0 resting and G1 (cycling) cell cycle phases been objectively characterized, but a separate subpopulation of quiescent cells that are functionally viable and deeply committed to nonproliferation, the Q cells, has been identified. Multiparameter cytofluorimetric analysis, methyl14C-thymidine incorporation, automated image analysis, and mitogen stimulation studies have shown that the "Q" cell, compared to the "G0" resting but easily recruitable cell, exhibits quite lower red and green AO emission, possesses 2c to 4c DNA content (rather than only 2c), has a higher average optical density, and is either nonrecruitable or recruitable-with-difficulty in PHA-stimulated lymphocyte cultures.     

Effects of fetal intravenous glucose challenge in normal and growth retarded fetuses

Fetal intravenous glucose challenge test (0.75 g/kg of estimated fetal weight) was performed at 26-33 weeks gestation in 9 patients undergoing fetal blood sampling (FBS) by ultrasound guided needling from the umbilical vein. The indication for FBS was rapid karyotyping for fetal malformations in 5 (control group) and severe intrauterine growth retardation in the remaining 4 (IUGR group). Fetal blood samples were taken before the glucose infusion and after 1, 3, 5, 10 and 15 min; glucose and insulin were assayed on each occasion and acid-base balance at 0 and 5 min. Basal fetal pO2, pH, glucose and insulin were lower in the IUGR group than in controls. Following the glucose challenge, fetal glucose levels were similar in the two groups, but in the IUGR group the latter part of the glucose curve was characterized by a slower and delayed return to basal levels. In control fetuses the insulin response following the glucose challenge peaked at 3 min while in IUGR no change in insulin concentration was detected. Fetal pO2 did not change in either group; the median change in fetal pH was significantly different between the two groups (controls: +0.01; IUGR: -0.04; P less than 0.05) and there was a significant correlation between basal pO2 and the change in fetal pH (r = 0.79) (P less than 0.02). These results support the concept of a low energy state in IUGR. Fetal glucose supplementation in IUGR is unlikely to be of benefit and may even exacerbate underlying acidosis.     

Studies on the possible biological effects of 50 Hz electric and/or magnetic fields: Evaluation of some glycolytic enzymes,glycolytic flux,energy and oxido-reductive potentials in human erythrocytes exposed in vitro to power frequency fields

An attempt has been made to understand whether 50 Hz electric and magnetic fields (EMFs) are involved in producing bioeffects by exposing human erythrocytes in vitro. The study evaluated some key glycolytic enzymes, glucose consumption, lactate production, energy charge, 2,3-diphosphoglycerate, and reduced glutathione levels. all of which are biochemical parameters significant to erythrocyte function. Cells exposed to individual or superimposed EMFs have not shown any significant difference compared with the controls. © 1993 Wiley-Liss. Inc.